Summary: Endogenous protease inhibitors of several classes can regulate the turnover of the extracellular matrix associated with loss of tissue homeostasis and formation of the tumor microenvironment. The tissue inhibitors of metalloproteinases (TIMPs) are the most specific and well-studied protease inhibitors for the enzymes associated with establishment and progression of the tumor microenvironment. The TIMPs are a family of four small highly conserved proteins. The TIMPs have been identified in species ranging from drosophila, zebra fish and C. elegans to humans, suggesting that these proteins are ancient eukaryotic proteins. The demonstration that TIMP 1 and TIMP-2 inhibited tumor cell invasion and metastasis in mouse xenograft models in the early 1990s, spurred a significant effort on the part of the pharmaceutical industry to develop synthetic MMP inhibitors as potential new cancer therapeutics. Unfortunately, these synthetic MMP inhibitors failed in all subsequent human clinical trials. Although many hypotheses have been put forward to explain the failure of these drugs in human cancer patients, the true nature of this failure remains unknown. The mammalian TIMP family has four members, which share significant homology and structural identity at the protein level. TIMP-2 is unique as a member of the TIMP family in that in addition to inhibiting MMPs TIMP-2 selectively interacts with MT1-MMP to facilitate the cell-surface activation of pro-MMP-2. Thus, TIMP-2 functions both as an inhibitor of MMPs, and is required for the cellular mechanism of pro-MMP-2 activation. TIMP-2 also has a distinct gene structure compared with the other three members of the TIMP family. An interesting relationship exists between the TIMPs and the synapsin gene family in that three members of the TIMP family are nested within the synapsin genes. The synapsin 1 gene nests TIMP-1, synapsin 2 nests TIMP-4 and synapsin 3 nests TIMP-3. TIMP-2 is the only member of the TIMP family that is not nested within a gene of the synapsin family. The synapsin-TIMP gene nesting relationship began phylogenetically as far back as Drosophila. A recent report describes a nested gene within the very large (60 kb) first intron of the TIMP-2 gene, known as differential display clone 8 (DDC8), which at first was thought to encode a testis specific protein. Furthermore, it has been shown that the brain of the TIMP-2-deficient mouse generated by deletion of exon 1 contains TIMP-2 mRNA encoding exons 2-5 downstream of DDC8 sequence, suggesting alternative splicing between these two genes. We have recently demonstrated that exogenous and enhanced expression of endogenous TIMP-2 promotes the differentiation of human microvascular endothelial cells. Jaworski and colleagues have demonstrated that TIMP-2 inhibits neurite proliferation and promotes differentiation. TIMP-2 is frequently under expressed in tumors compared with normal tissue. These observations led us to postulate that re-expression of TIMP-2 in tumor cells and tumor xenografts may influence tumor progression, tumor cell differentiation, epithelial-to-mesenchymal transition and metastatic capacity. We have previously demonstrated that TIMP-2 suppressed the EGF-induced growth of the human lung cancer cell line A549. Preliminary evidence shows that endogenous expression of TIMP-2 in the A549 cell line is very low and the forced expression of TIMP-2 or Ala+TIMP-2 results in over 80 % suppression of tumor growth. We are now examining the mechanism of this effect with respect to the role of TIMP-2 in promoting tumor cell differentiation. In addition we are attempting to characterize the relative contributions of the metalloproteinase inhibitor activity, anti-angiogenic activity and possible direct anti-tumor effects of TIMP-2 to the overall potent anti-tumor activity of this protein